Pharmaceutical composition, substrate comprising a pharmaceutical composition, and use of a pharmaceutical composition

ABSTRACT

Use of a pharmaceutical composition for the local treatment or prevention of a tissue infection at an infection site, the pharmaceutical composition comprising at least two different antibiotics of group A or pharmaceutically acceptable derivatives thereof, or an antibiotic of group A and at least one antibiotic of group B or pharmaceutically acceptable derivatives thereof. Group A comprises primarily intracellular active antibiotics working as inhibitor of bacterial RNA polymerase; as inhibitor of gyrase; or as inhibitor of bacterial protein synthesis. Group B comprises primarily extracellular active antibiotics working as inhibitor of bacterial cell wall synthesis; or inhibitor of bacterial protein synthesis; or by direct destabilisation or rupture of the bacterial cell wall.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a National Phase Patent Application of InternationalPatent Application Number PCT/EP2008/006046, filed on Jul. 23, 2008,which claims priority of European Patent Application Number 07075639.0,filed on Jul. 23, 2007.

INTRODUCTION AND SUMMARY

The invention relates to the use of a pharmaceutical composition, apharmaceutical composition, a pharmaceutical composition for treatmentof extracellular and/or intracellular microbial infected cells, and asubstrate comprising a pharmaceutical composition. The invention relatesfurther to the use of antibiotics as anti-adhesives againstmicroorganisms on surfaces.

Infections of bone and tissue are the severest problem of orthopaedicsand surgery, in particular due to increasing operation frequency. 30% ofall bone infections become chronic despite of treatment. Further, manycases are known in which an infection reoccurred after allegedsuccessful earlier treatment. In 3% of all cases, amputation is the onlyremaining option. Systemic treatment with antibiotics is difficult sinceantibiotics penetrate through bone generally only very poorly and thusconcentrations being sufficiently high to eliminate an infection arehardly achievable.

Local application of antibiotics is better suited for therapy ofinfections of bone and other tissue than systemic antibiotic therapysince by local application higher concentrations of antibiotics can beachieved at the treatment site than by systemic application. Aprerequisite for a successful local antibiotics therapy is a precedingradical surgical therapy, including debridement of all bone or tissuenecroses and excision of all foreign material. Local antibiotics carrierknown from prior art are bone cements made from polymethylmethacrylate(PMMA), beads made from PMMA, collagen fleeces and bone substituents.These carriers are commercially available with a limited number ofantibiotics applied onto them: gentamicin, tobramycin, clindamycin,vancomycin and teicoplanin.

Though local antibiotics therapy employing the above-mentionedantibiotics have already improved treatment of bone and jointinfections, such therapy fails in a significant number of cases (up to16%). Therapy failure, however, often finally leads to the necessity ofan amputation.

The major reasons for therapy failure are a) resistances against certainantibiotics, b) ineffectiveness of antibiotics against sessile bacteria,c) intracellular localised bacteria and d) induction of Small ColonyVariants. In this context, ineffectiveness according to item b) is dueto biofilm formation and cessation of proliferation of the bacteria tobe eliminated. Further, in this context, intracellular is to beunderstood with respect to a cell of the host, i.e. of the subject to betreated. Thus, if bacteria are inside a cell of the host, antibioticsbeing unable to penetrate to the inner of the cell cannot act on thebacteria to be eliminated.

It has been known for some time that Staphylococci can survive insideleucocytes. Further, it is known that strains of Staphylococcus aureusshowing the so-called Small Colony Variant phenotype can be internalisedby keratinocytes and endothelial cells and can persist intracellularly.It was demonstrated that they remain intracellularly inside lysosomes.S. aurerus of normal phenotype can also be internalised by endothelialcells, fibroblasts and keratinocytes and can remain intracellularlyinside lysosomes.

It was demonstrated that S. aurerus isolates display a dichotomy:Whereas cytotoxic strains survive in keratinocytes and fibroblasts andinduce significant cytotoxicity to their host cells due to intracellulardivision, non-cytotoxic strains are killed inside of keratinocytes andfibroblasts, indicating that uptake of S. aurerus represents animportant mechanism of cell-autonomous host defence (Krut, O. et al.,Infection and Immunity, 2003, 71: 2716-2723).

S. aurerus can also be internalized by osteoblasts, but osteoblastscannot kill internalized non-cytotoxic staphylococci, instead they canpersist over days and weeks inside osteoblasts without proliferation.After lysis of the osteoblasts, the Staphylococci can proliferate again.Intracellular persistence of Staphylococci and possibly other bacteriain osteoblasts and their potential to persist intracellularly insidelysosomes may play a particular role when looking at bone infections.This could be causative for chronic progression of bone infections.

Though it is still not known exactly whether pseudomonades, streptococciand enterococci can persist in osteoblasts, intracellular persistence ofthese bacteria could be shown in general. This intracellular persistencewas hitherto only thought to be possibly related with chronicprogression of other diseases, but since pseudomonades, streptococci andenterococci are frequent pathogens with respect to bone infections,their intracellular persistence might be causative for chronicprogression of bone infections.

Based on this assumption, it is explainable why allegedly successfullytreated bone infections can re-outbreak even after years. Inside a hostcell the bacteria are protected against numerous antibiotics whichcannot penetrate the cell membrane (e. g. penicillins, glycopeptides).Though an acute infection being induced by planktonic (floating)bacteria might be treated with these antibiotics, bacteria can remainintracellularly and cause a re-infection after release from the hostcell.

In prior art, bone and soft tissue infections are treated locally mainlywith aminoglycosides (gentamicin, tobramycin) which usually cannotpenetrate the cell membrane of host cells. On the other hand, it wasreported that aminoglycosides can accumulate in lysosomes of fibroblastsbut are inactive due to the low pH of lysosomes.

Consequently, antibiotics used in prior art for local therapy ofinfections of bone and other tissue are not suited to treat all theseinfections successfully. In particular, local antibiotics carriercontaining only gentamicin are ineffective against infections ofbacteria showing a Small Colony Variant phenotype and can even induceformation of Small Colony Variant phenotypes. None of the antibioticsused at present for local therapy of infection can eliminateintracellular localized bacteria.

WO 2006/064517 discloses an antibiotic composition comprising a firstantibiotic inhibiting the bacterial protein synthesis and a secondantibiotic not inhibiting the bacterial protein synthesis.

U.S. Pat. No. 5,217,493 discloses an implantable medical device which iscoated against biofilm colonization with rifampin and novobiocin, orrifampin and minocycline.

Given an embodiment it is possible to provide a pharmaceuticalcomposition for treating and preventing extra- and intracellularinfections of cells, especially tissue cells, a substrate carrying suchcomposition, and a method of applying such a composition and substrate.

It is further possible to decrease the adhesion rate of microorganismson different substrate surfaces.

In an embodiment of the invention, the local treatment and prevention isdone at an infection site. The tissue to be treated can e.g. be softtissue and/or bone tissue including what is generally denoted as “bone”.The pharmaceutical composition comprises at least two differentantibiotics of group A or pharmaceutically acceptable derivativesthereof, or an antibiotic of group A and at least one antibiotic ofgroup B or respective pharmaceutically acceptable derivatives thereof.Group A comprises primarily intracellular active antibiotics working asinhibitor of bacterial RNA polymerase, as inhibitor of gyrase or asinhibitor of bacterial protein synthesis. Group B comprises primarilyextracellular active antibiotics working as inhibitor of bacterial cellwall synthesis or as inhibitor of bacterial protein synthesis ordestabilise or rupture the bacterial cell wall directly.

In the context of the present description a tissue infection isunderstood as an extracellular and intracellular infection of tissuecells caused by microorganisms.

In order to circumvent resistances against the antibiotics used,particularly in long-term treatments, a combination of at least twoantibiotics can be chosen. Such a combination results also in higherefficacy. Though it is generally considerable to use only intracellularactive antibiotics, a combination of an intracellular active antibiotic(group A) with an extracellular active antibiotic (group B) may also bechosen. Though antibiotics of group B are not intracellular active, theycan inhibit formation of resistances since they act on extracellularbacteria in a bactericidal manner and resistances are only formed inplanktonic, proliferating populations of bacteria. Since theextracellular active antibiotics of group B show a different mechanismof action than the antibiotics of group A, parallel resistances canhardly occur.

The pharmaceutical composition to be used can comprise furtheradditives, dispersants, solvents or carrier substances etc. known perse.

In order to achieve good results in treating infections of bone andother tissue, at least one of the antibiotics chosen should, in anembodiment, fulfil at least one of the following criteria:

-   a) It should penetrate the cell membrane of the host cell (i. e. the    cell of the subject to be treated inside which the bacteria to be    eliminated are located).-   b) It should be able to reach the inside of the lysosomes of the    host cell.-   c) It should be active at low pH (particularly at that pH being    present in lysosomes, i. e. ca. pH 4 to pH 5).-   d) It should have a bactericidal activity.-   e) It should show its bactericidal activity also against    non-proliferating bacteria.

In an embodiment at least one of the antibiotics chosen fulfils aplurality of the criteria mentioned above. In another embodiment,fulfilment of all of these criteria is achieved. In still anotherembodiment, a fulfilment of all criteria by all antibiotics chosen isachieved.

In an embodiment said antibiotics of group A working as inhibitor ofbacterial RNA polymerase comprise ansamycins, particularly rifamycins.Particularly, rifampin, rifabutin, rifapentine or rifamixin may bechosen. A pharmaceutical composition containing rifampin is particularlysuited in eliminating intracellular Staphylococci, which were shown tobe eliminated within 3 days after local administration of an accordingpharmaceutical composition.

In a further embodiment said antibiotics of group A working as inhibitorof gyrase comprise fluoroquinolones. The fluoroquinolone moxifloxacin isparticularly chosen.

In an embodiment said antibiotics of group A working as inhibitor ofbacterial protein synthesis comprise streptogramins like, e. g.,quinupristin or dalfopristin. In an embodiment, a combination ofquinupristin and dalfopristin is used. It is to be noted that thepharmaceutical composition to be used may contain more than a singleantibiotic of each group (and more than two antibiotics of group A if noantibiotic of group B is used) and thus more than two antibiotics intotal.

In an embodiment said antibiotics of group B working as inhibitor ofbacterial cell wall synthesis or destabilising and rupturing the cellwall directly comprise glycopeptides, fosfomycin and polypeptides. In anembodiment the glycopeptides chosen are vancomycin and teicoplanin. Inthe same or another embodiment the polypeptides chosen are bacitracin,polymyxin B as well as other polymyxins and daptomycin.

In an embodiment said antibiotics of group B working as inhibitor ofbacterial protein synthesis comprise aminoglycosides. In this context,particularly arbekacin may be chosen.

An exemplary pharmaceutical composition to be used comprises a rifamycinand an aminoglycoside. Another exemplary pharmaceutical compositioncomprises rifampin and arbekacin; such a composition essentially coversthe entire germ spectrum to be eliminated and is effective againstproblematic bacteria like methicillin-resistant S. aurerus (MRSA) ormethicillin-resistant S.epidermidis (MRSE). Both antibiotics areeffective also against non-proliferating (resting) bacteria and aretemperature resistant (heat stable) so that they can be added to a bonecement made of poly(methylmethacrylate) (PMMA), to PMMA bead chains, andto spacers for revision operations.

Another pharmaceutical composition to be used comprises a rifamycin andfosfomycin. Still another pharmaceutical composition comprises rifampinand fosfomycin; such a composition also essentially covers the entiregerm spectrum to be eliminated and is also effective against problematicbacteria like MRSA and MRSE. Fosfomycin has the further property that itbinds reversibly to hydroxyl apatite and thus remains, even afterrelease from a carrier, longer in a bone than other antibiotics.Further, fosfomycin is the smallest antibiotic known and diffuses orpenetrates very well through or into bone tissue.

A further pharmaceutical composition to be used comprises a rifamycinand a fluoroquinolone. Another pharmaceutical composition comprisesrifampin and moxifloxacin.

One object is also addressed by providing a pharmaceutical composition.Such a pharmaceutical composition can be used for the local treatmentand prevention of a tissue infection at an infection site, wherebyfurther embodiments of such a use are analogous to those explained aboveand to which in entirety reference is made hereby.

Such a pharmaceutical composition comprises at least two differentantibiotics of group A′ or pharmaceutically acceptable derivativesthereof, or an antibiotic of group A′ and an antibiotic of group B′ orpharmaceutically acceptable derivatives thereof. In this case, group A′comprises the primarily intracellular active antibiotics ansamycins,particularly rifamycins such as rifampin, rifabutin, rifapentine orrifamixin; fluoroquinolones, particularly moxifloxacin; streptogramins,particularly quinupristin and/or dalfopristin. Group B′ comprises theprimarily extracellular active antibiotics glycopeptides, particularlyvancomycin or teicoplanin; fosfomycin; polypeptides, particularlybacitracin, daptomycin, or polymyxin B; and aminoglycosides,particularly arbekacin. It is to be noted that glycopeptides cannot bethe second antibiotic of a pharmaceutical composition comprising onlytwo antibiotics and comprising an ansamycin as first antibiotic.

In an embodiment the pharmaceutical composition comprises only aglycopeptide, a polypeptide or fosfomycin as possible antibiotic ofgroup B′, but no aminoglycosides are used as antibiotic of group B′. Inanother embodiment no streptogramins are used as antibiotic of group A′.

In an embodiment the antibiotics are chosen in such a way that eithernone or all antibiotics in the pharmaceutical composition work asinhibitors of protein synthesis, i.e. either a) only differentstreptogramins, or a streptogramin and an aminoglycoside may be used orb) no streptogramins and no aminoglycosides may be used at all.

In an alternative embodiment the pharmaceutical composition comprises arifamycin and an aminoglycoside, particularly rifampin and arbekacin.

In another embodiment the pharmaceutical composition comprises arifamycin and fosfomycin, particularly rifampin and fosfomycin.

Such a composition comprises rifamycin and fosmycin in such aconcentration that the rifamycin reaches a concentration of 0.005 to 100μg/ml, preferably 0.006 to 80 μg/ml, most preferably 0.0075 to 20 μg/mlat the site to be treated. Fosfomycin reaches a concentration of 1 to1000 μg/ml, preferably 5 to 800 μg/ml, most preferably 10 to 200 μg/mlat the site to be treated.

In yet another embodiment the pharmaceutical composition comprises arifamycin and polypeptide, particularly rifampin and daptomycin.

Such a composition comprises rifamycin and daptomycin in such aconcentration that the rifamycin reaches a concentration of 0.005 to 100μg/ml, preferably 0.006 to 80 μg/ml, most preferably 0.0075 to 20 μg/mlat the site to be treated. Daptomycin reaches a concentration of 01 to100 μg/ml, preferably 0.5 to 80 μg/ml, most preferably 1 to 20 μg/ml atthe site to be treated.

In still another embodiment the pharmaceutical composition comprises arifamycin and a fluoroquinolone, particularly rifampin and moxifloxacin.

One object is also achieved by a pharmaceutical composition for thetreatment of extracellular and/or intracellular microbial infected cellsand/or for the prevention of microbial infections of cells comprising atleast one antibiotic acting as an inhibitor of bacterial RNA polymerase,at least one antibiotic affecting the bacterial cell wall or itssynthesis, and/or at least one antibiotic acting as a gyrase inhibitor.

The treatment preferably occurs locally or systemically.

Advantageously, ansamycins, particulary rifamycins such as rifampin,rifabutin, rifapentine or rifamixin are used as inhibitors of bacterialRNA polymerase. As antibiotics affecting the bacterial cell wall or itssynthesis glycopeptides, particularly vancomycin or teicoplanin,fosfomycin and polypeptides, particularly bacitracin and daptomycin arechosen. As a gyrase-inhibitors fluoroquinolones, particularlymoxifloxacin, is applied.

Rifamycin is used in concentrations between 0.005 to 100 μg/ml,preferably 0.006 to 80 μg/ml, most preferably 0.0075 to 20 μg/ml.Fosfomycin is used in concentrations of 1 to 1000 μg/ml, preferably 5 to800 μg/ml, most preferably 10 to 200 μg/ml. Moxifloxacin is applied in aconcentration between 0.1 to 500 μg/ml, preferably 0.5 to 200 μg/ml,most preferably 1 to 100 μg/ml. Daptomycin is used in concentrations of0.1 to 100 μg/ml, preferably 0.5 to 80 μg/ml, most preferably 1 to 20μg/ml. The same concentrations are preferably used in a combination ofrifamycin, fosfomycin, daptomycin and/or moxifloxacin.

The pharmaceutical composition is especially effective in case ofinfected cells such as osteoblasts, leucocytes, erythrocytes,keratinocytes, fibroblasts, fat cells, muscle cells and/or endothelialcells.

Furthermore, the pharmaceutical composition is effective againstmicrobial infection caused by gram-negative and/or gram-positivebacteria, preferably by the Staphyloccoci type, most preferably byStaphylococcus aureus.

In an embodiment the pharmaceutical compositions to be used furthercomprise a biofilm formation inhibitor. Every substance reducing orinhibiting at least partially the attachment of germs, especiallybacteria on a surface or the ability of germs to accumulate on a surfaceto form a biofilm on that surface is considered as biofilm formationinhibitor.

In an embodiment salicylic acid or a pharmaceutical active derivative orsalt thereof is used as biofilm formation inhibitor. Particularly, acombination of salicylic acid and an aminoglycoside may be used.Salicylic acid enhances the microbial activity of aminoglycosidesagainst bacteria, especially against E. coli and Klebsiella pneumoniae:Salicylates enter a cell in a protonated form, thereby increasing themembrane potential of the cell. This, in turn, simplifies the uptake ofaminoglycosides into the interior of the cell.

Even salicylic acid itself shows an effect on bacteria. Growth ofencapsulated Klebsiella pneumoniae in the presence of salicylate resultsin reduced synthesis of capsular polysaccharides. The loss of capsularmaterial exposes the cell surface of K. pneumoniae to the host defencemechanisms, thus shortening the time required for infection clearance.Salicylic acid reduces the ability of bacteria to adhere onto surfacesand to form biofilms. Though salicylic acid does not provide 100%protection against biofilm formation, it supports the effect ofantibiotics.

Acetylsalicylic acid and/or its predominant metabolite salicylic acidexhibit definable impacts both in vitro and in vivo on microbialvirulence phenotypes. Bacterial virulence factors help mediate infectionby bacteria in a host organism. The following effects have been noted:reduction of adhesion to relevant biomatrices, reduction of capsuleproduction, mitigation of biofilm formation, and diminution ofvegetation growth, intravegetation bacterial proliferation, andhematogenous dissemination in experimental infective endocarditis.Salicylic acid also regulates positively the translation of specificgene loci including multiple antibiotic-resistance loci. Further, itinduces cytoplasmic proteins; and increases quinolone resistance.

The synthesis of some types of fimbriae in E. coli e.g. colonizationfactor antigen, P fimbriae and type 1 fimbriae are reduced followinggrowth in the presence of salicylate. Because fimbriae play a criticalrole in the attachment of E. coli to epithelial surfaces, salicylatetreatment might prevent infection caused by some strains of fimbriatedE. coli. Salicylate also limits adherence of E. coli to silasticcatheters.

Chemotaxis in bacteria is modulated through regulation of flagellarotation. This rotation, when counterclockwise, leads to swimming alonga linear trajectory and, when clockwise, leads to tumbling. Salicylateis recognized as a chemorepellant by the E. coli tsr gene product. Thisrecognition leads to prolonged tumbling of motile E. coli and ultimatelycauses cells to migrate away from salicylate. Swarming behaviour of E.coli is also inhibited by salicylate in a concentration-dependentmanner. Production of the flagellum itself in E. coli is inhibited bygrowth in the presence of salicylate. This is mediated by inhibiting theproduction of flagellin, the protein monomer constituting the flagella.It has also been speculated that inhibition of flagella synthesis andmotility in E. coli by salicylate is due to reduced synthesis in OmpFsynthesis, which may be required for flagella assembly.

Biofilms consist of microorganisms and other matter encased in apolysaccharide matrix of microbial origin. Growth of Pseudomonasaeruginosa and Staphylococcus epidermidis in the presence of salicylatereduces the production of extracellular polysaccharide required forbiofilm formation. The reduction in biofilm formation decreases theability of these organisms to adhere to contact lenses and medicalpolymers. A component of biofilm production in S. epidermidis isextracellular slime which is composed of a complex mixture ofpolysaccharides, teichoic acids and proteins. Production ofslime-associated proteins and teichoic acids is inhibited in S.epidermidis by salicylate.

In case of S. aurerus, salicylic acid mitigates two distinct virulencephenotypes that are of key relevance for matrix binding, i.e. tofibrinogen and fibronectin, and α-hemolysin activity. These effects arespecifically associated with salicylic acid-mediated reduction in theexpression of the respective structural genes, i.e., fnbA, fnbB, andhla. In addition to the suppression of matrix protein binding andcytolytic profiles, enhanced exoenzyme and protein A production occursin the presence of salicylic acid. These findings raise the likelihoodthat salicylic acid executed its antimicrobial effects through one ormore global regulatory networks rather than a decrease in general genetranscription. Global regulon sarA and the global regulon agr aremitigated by salicylic acid, corresponding to the reduced expression inof the hla and fnbA genes in vitro. It should be noted that S. aurerusvirulence parameters were not completely suppressed by salicylic acidbut were reduced, in a drug concentration-dependent manner, by a maximumof approximately 50%.

In an embodiment the infected tissue to be treated is acutely orchronically infected. A combination of an acute and a chronic infection,i.e. the acute infection overlying the chronic infection, might also betreated.

The object is also achieved by providing a substrate for medicalpurposes according to claim 21. The substrate is preferably used ascarrier of the pharmaceutical composition when locally treating andpreventing the tissue infection. In a further embodiment the substrateis also used locally after removal of the infected tissue as asupplement in surgical debridement.

In one embodiment the substrate can be soaked with the pharmaceuticalcomposition to be used. In another embodiment the pharmaceuticalcomposition can be dispersed in a base material of the substrate. Instill another embodiment, the pharmaceutical composition can bepolymerised with the base material. Thus, it is possible to coat thesubstrate with the pharmaceutical composition and/or to incorporate thepharmaceutical composition into the substrate.

In a preferred embodiment the substrate underwent special treatment e.g.sand blasting or hydroxyl apatite coating before the pharmaceuticalcomposition is applied.

Within the scope of the present description is also a coating made of asupport material in which the pharmaceutical composition is present e.g.in a dispersed form. Such support material can include polylactides. Thesupport material with the dispersed pharmaceutical composition is thenapplied as a coating onto the substrate—either directly onto the surfaceof the latter or onto a layer being already present on that surface oron another layer.

In an embodiment the substrate comprises a fleece, a fabric, a polyethyl methacrylate, a copolymer of methylmethacrylate andmethylacrylate, a resorbable polymer, polyethylene, a metal or a metalalloy e.g. a Ti6Al4V alloy or another titaniumium alloy, a ceramic, abone cement, particularly made from a polymeric material or from calciumphosphate and/or a bone substitute. Thus, PMMA bead chains consistingmainly of a copolymer of methylmethacrylate and methylacrylate as wellas glycine and a specific pharmaceutical composition to be administeredas local antibiotics carrier are a possible substrate. Further, the bonecement may be intended to be used for spacer and for revisionoperations.

In case of PMMA bead chains, the following mode of use is possible:firstly, the pharmaceutical composition is dispersed within the PMMAbase material. The powder is heated to 180° C. and filled into forms byinjection moulding. The pharmaceutical composition is being distributedall over the base material and can diffuse from the inner parts of aPMMA bead towards the surface, where it may interact with bacteria beingpresent around the PMMA bead chain. The PMMA bead chains may comprise0.1-10 wt %, preferably 0.5-8 wt %, most preferably 1-5 wt %antibiotic(s).

In another embodiment, particularly in case of revision operations, thesubstrate is an implantable prosthesis, wherein joint prostheses andparticularly knee, hip, shoulder, elbow prostheses as well as vertebralimplants are respective examples. Furthermore, all implants for traumasurgery like screws, plate, etc. may be used as substrate. The substratecoating may comprise 10-1000 μg/cm², preferably 20-500 μg/cm², mostpreferably 50-300 μg/cm² antibiotic(s) per cm² substrate surface area.

In an embodiment the fleece or fabric comprises a natural or syntheticfibre, which can be biodegradable, wherein polylactide (polylactic acid)is an exemplary material. In another embodiment the fleece or fabriccomprises collagen, wherein the fleece may consist essentially ofcollagen. In the latter case, the collagen fleece is also completelybiodegradable. The fleece may comprise 0.01-10 mg/cm², preferably 0.1-8mg/cm², most preferably 0.5-5 mg/cm² antibiotic(s) per cm² fleece.

Further a method for locally treating a subject with a pharmaceuticalcomposition is described, the pharmaceutical composition comprising:

-   -   at least two different antibiotics of group A or        pharmaceutically acceptable derivatives thereof or    -   an antibiotic of group A and at least one antibiotic of group B        or pharmaceutically acceptable derivatives thereof, wherein    -   group A comprises intracellular active antibiotics working as    -   inhibitor of bacterial RNA polymerase,    -   inhibitor of gyrase or    -   inhibitor of bacterial protein synthesis and group B comprises        extracellular active antibiotics working    -   as inhibitor of bacterial cell wall synthesis,    -   as inhibitor of bacterial protein synthesis or    -   by direct destabilisation or rupture of the bacterial cell wall.

This method may be particularly used for treating a tissue infection ofsaid subject, wherein the tissue may be, e.g., soft tissue and/or bonetissue and/or bone. These infections might occur due to a surgicaloperation, particularly due to an operation related to implanting animplant into a human or non-human body. Thus, the treatment might beapplied to a human or non-human body.

With respect to further embodiments of this aspect reference is made theexplanations given above which are analogously applicable for saidmethod, particularly regarding the substrate to be used and theantibiotics to be chosen.

A second object is achieved by using a combination of at leastantibiotic acting as an inhibitor of bacterial RNA polymerase and atleast one antibiotic affecting the bacterial cell wall or its synthesisas anti-adhesives against microorganisms on surfaces.

The inhibitor of bacterial RNA polymerase is preferably selected fromthe group comprising ansamycins, particulary rifamycins such asrifampin, rifabutin, rifapentine or rifamixin.

The antibiotic affecting the bacterial cell wall or its synthesis ispreferably selected from the group comprising glycopeptides,particularly vancomycin or teicoplanin, fosfomycin and polypeptides,particularly bacitracin or daptomycin. A preferred combination comprisesrifamycin and fosfomycin.

In a further embodiment the microorganisms are gram-negative and/orgram-positive bacteria, preferably of the Staphyloccoci type, mostpreferably Staphylococcus aureus.

The combination of the at least one inhibitor of bacterial RNApolymerase and the at least one antibiotic affecting the bacterial cellwall or its synthesis is preferably attached or coated onto surfacesmade of metal, preferably titanium, steel or metal alloy, ceramics, andbone cement or hydroxyl apatite.

When coated on a substrate the combination may comprise rifamycin andfosfomycin in a concentration between 10 and 1000 μg/cm², preferably 20to 500 μg/cm², most preferably 50-200 μg/cm², respectively.

Advantageously, the antiadhesive effect is accompanied by a bactericidaleffect on the tissue surrounding the coated surfaces.

BRIEF DESCRIPTION OF THE DRAWINGS

Examples of embodiments are explained in further detail by means of thefollowing figures and examples.

FIG. 1 shows CFU of S. aurerus ATTC 6538P in cell culture supernatant ofosteoblastic MG63 cells.

FIG. 2 shows CFU of S. aurerus BAA44 in cell culture supernatant ofosteoblastic G63 cells.

FIG. 3a shows metabolic activity of osteoblastic MG63 cells afterinfection with S. aureus ATTC 6538P followed by addition of rifampin tothe cell culture supernatant after treatment with lysostaphin to removeextracellular bacteria.

FIG. 3b shows metabolic activity of osteoblastic MG 63 cells afterinfection with S. aureus ATTC 6538P followed by addition of fosfomycinto the cell culture supernatant after treatment with lysostaphin toremove extracellular bacteria.

FIG. 3c shows metabolic activity of osteoblastic MG63 cells afterinfection with S. aureus ATTC 6538P followed by addition of fosfomycin,rifampin and their combination to the cell culture supernatant aftertreatment with lysostaphin to remove extracellular bacteria.

FIG. 3d shows metabolic activity of osteoblastic MG63 cells alterinfection with S. aureus ATTC 6538P followed by addition of after addinga mixture containing 10 μg/ml fosfomycin and 0.006-0.0075 μg/ml rifampinto the cell culture supernatant after treatment with lysostaphin toremove extracellular bacteria.

FIG. 3e shows metabolic activity of osteoblastic MG63 cells afterinfection with S. aureus ATTC 6538P followed by addition of moxifloxacinto the cell culture supernatant after treatment with lysostaphin toremove extracellular bacteria.

FIG. 4a shows CFU of S. aurerus BAA44 located in osteoblastic MG63 cellsafter adding fosfomycin to the cell culture supernatant of osteoblasticMG63 cells after treatment with lysostaphin to remove extracellularbacteria.

FIG. 4b shows CFU of S. aurerus BAA44 located in osteoblastic MG63 cellsafter adding rifampin to the cell culture supernatant of osteoblasticMG63 cells after treatment with lysostaphin to remove extracellularbacteria.

FIG. 4c shows CFU of S. aurerus BAA44 located in osteoblastic MG63 cellsafter adding a mixture containing 50 μg/ml fosfomycin and 2.5-40 μg/mlrifampin to the cell culture supernatant of osteoblastic MG63 cellsafter treatment with lysostaphin to remove extracellular bacteria.

FIG. 4d : shows CFU of S. aurerus BAA44 located in osteoblastic MG63cells after adding a mixture containing 25-400 μg/ml fosfomycin and 2.5μg/ml rifampin to the cell culture supernatant of osteoblastic MG63cells after treatment with lysostaphin to remove extracelluar bacteria.

FIG. 4e shows CFU of S. aurerus BAA44 located in osteoblastic MG63 cellsafter adding fosfomycin, rifampin and a mixture containing 50 μg/mlfosfomycin and 10 μg/ml rifampin to the cell culture supernatant ofosteoblastic MG63 cells after treatment with lysostaphin to removeextracellular bacteria.

FIG. 4f shows CFU of S. aurerus BAA44 located in osteoblastic MG63 cellsafter adding fosfomycin, rifampin and a mixture containing 25 μg/mlfosfomycin and 2.5 μg ml rifampin to the cell culture supernatant ofosteoblastic MG63 cells after treatment with lysostaphin to removeextracellular bacteria.

FIG. 4g : shows CFU of S. aurerus BAA44 located in osteoblastic MG63cells after adding fosfomycin, rifampin and a mixture containing 50μg/ml fosfomycin and 20 μg/ml rifampin to the cell culture supernatantof osteoblastic MG63 cells after treatment with lysostaphin to removeextracellular bacteria.

FIG. 5a : shows CFU of S. aurerus ATTC 6538P per titanium disc after 1.5h incubation of S. aurerus on vancomycin or rifampin/fosfomycin coatedtitanium disc, which were either washed once or thrice beforeincubation.

FIG. 5b : shows CFU of S.epidermidis ATTC 35984 per titanium disc after2 h incubation with S.epidermis on rifampin/fosfomycin coated titaniumdiscs, which were washed twice before incubation.

FIG. 5c : shows CFU of S. aurerus BAA44 per titanium disc after 1.5 hincubation of with S. aurerus on vancomycin or rifampin/fosfomycincoated titanium discs, which were washed either once or twice beforeincubation.

FIG. 5d : shows CFU of S. aurerus BAA44 on titanium discs or in thesupernatant after overnight incubation of S. aurerus with vancomycin orrifampin/fosfomycin coated titanium discs in Minimal Medium.

FIG. 6: shows CFU of in osteoblastic MG63 cells intracellular located orto osteoblastic MG63 cells adhered S. aurerus BAA44 after incubationovernight with S. aurerus BAA44 and antibiotics.

DETAILED DESCRIPTION

1. Use of Rifampin and Fosfomycin or their Combination for the Treatmentof Extracellular Infections

1.1. Use of Rifampin and Fosfomycin or their Combination for theTreatment of Extracellular Infections of Osteoblastic MG63 CellsInfected with Staphylococcus aureus Subsp. aureus Rosenbach (ATTC 6538P)

Osteoblastic MG63 cells were detached with the cell detachment mediumAccutase 24 hours before infection. The cell number was determined usingthe Neubauer counting chamber. Cells were seeded onto uncoated 24 wellplates with a cell density of 1.5×10⁴ cells/cm² in 1 ml DMEM (Dulbecco'sModified Eagle's Medium) with 10% FOS (fetal calf serum), 1% Glutamax-1and 1% Natrium Pyruvat and incubated at 37° C. and 5% CO₂.

An overnight culture of S. aurerus ATTC 6538P was prepared by infecting5 ml Caso-Bouillon medium with S. aurerus ATTC 6538P. The cultures wereincubated with shaking (450 U/min) over night at 37° C. 100 μl of theovernight cultures were transferred into 5 ml Caso Boulillon medium andincubated for 2 h at 37° C. with shaking (450 U/min) prior to infection.

The cell culture supernatant of the osteoblastic MG63 cells was removedwith a pipette from the wells. 1 ml containing 1×10⁶ S. aurerus ATTC6538P cells was added to each well. Two 24 well plates were incubatedwith S. aurerus ATTC 6538P. The combined osteoblastic cells and bacteriawere incubated for 1.5 h at 37° C. under 5% CO₂ atmosphere. The presenceof bacteria was determined using a microscope.

After 1.5 h the supernatant was removed and the wells were carefullywashed twice with 37° C. warm DMEM without additives. It wasmicroscopically checked, if not too many cells were detached during thewashing procedure. During the washing only the planktonic cells wereremoved, bacteria adhered to cells and the cell culture plastics werevisible in great numbers. Afterwards 1 ml of cell culture completemedium was added to each well containing following antibiotics:

-   -   100 μg/ml gentamicin    -   1 μg/ml rifampin    -   100 μg/ml fosfomycin disodium    -   1 μg/ml rifampin+100 μg/ml fosfomycin disodium    -   1 μg/ml rifampin+100 μg/ml gentamicin        No negative control without antibiotics was used since the        strong bacterial growth in absence of antibiotics would damage        the osteoblastic cells.

After incubation for 24 h, 100 μl of the cell culture supernatant wasstreaked out on Caso agar plates (Casein-peptone soymeal-peptone broth)directly, e.g. in case of rifampin, fosfomycin, rifampin/fosfomycin andrifmpicin/gentamicin, or after appropriate dilution, e.g. 1:100 in caseof gentamicin, and incubated overnight at 37° C. The supernatant of twowells per group was streaked out.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

The CFU (colony forming unit) of the supernatant was determined and isshown in FIG. 1.

FIG. 1 shows clearly a sensitivity of S. aurerus ATTC 6538P located inthe culture supernatant of osteoblastic MG63 cells towards the differentantibiotics with the exception of gentamicin Although the usedconcentrations were high compared to the MIC values (minimal inhibitoryconcentration) determined for this S. aurerus strain, the bacteria couldnot be removed completely with the antibiotic treatment. This is due tothe fact that the bacteria settled on the surface of the cells or thecell culture plastic, which results in reduced sensitivity toantibiotics. This simulates the in vivo situation where staphylococcireadily bind to the extracellular matrix and foreign bodies. The effectof rifampin, fosfomycin and the combination of rifampin/gentamicin ismoderate, whereas the combination rifampin/fosfomycin shows a strong,synergistic effect.

1.2. Use of Rifampin and Fosfomycin or their Combination for theTreatment of Extracellular Infections of Osteoblastic MG63 CellsInfected with Staphylococcus aureus Subsp. aureus (BAA44).

The experimental set up for the infection of osteoblastic MG63 cellsinfected with S. aurerus BAA44, a MRSA strain with additional resistanceagainst multiple antibiotics, was basically the same as above.

Following antibiotics were used:

-   -   100 μg/ml vancomycin    -   10 μg/ml rifampin    -   100 μg/ml fosfomycin    -   10 μg/ml rifampin+100 μg/ml fosfomycin    -   10 μg/ml rifampin+100 μg/ml vancomycin.

After incubation for 24 h 100 μl of the cell culture were streaked outon Caso agar plates (Casein-peptone soymeal-peptone broth) directly,e.g. in case of vancomycin, rifampin/fosfomycin and rifampin/vancomycin,or after appropriate dilution, e.g. 1:100 in case of rifampin andfosfomycin, and incubated overnight at 37° C. The supernatant of twowells per group was streaked out.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

The CFU (colony forming unit) of the supernatant was determined and isshown in FIG. 2.

FIG. 2 shows clearly a sensitivity of S. aurerus BAA44 located in theculture supernatant of osteoblastic MG63 cells towards the differentantibiotics. It is pointed out that it was necessary to adapt the CFUvalues logarithmical.

Rifampin in the used concentration shows as expected hardly anyefficacy, since S. aurerus BAA44 is a rifampin resistant strain. Alsothe antibiotic effect of fosfomycin is relatively small. However, thecombination rifampin/fosfomycin shows a strong, synergistic effect onextracellular S. aurerus BAA44, which was surprising and not expecteddue to the weak effect of the single compounds.

The effect of the combined rifampin/fosfomycin is even slightly betterthan the effect of vancomycin, which is one of the most importantantibiotics for the treatment of MRSA infections. The combination ofvancomycin and rifampin also shows a slight synergistic effect. It isnoteworthy that the concentration of vancomycin used in this experimentwas very high to increase the otherwise weak bactericidal effect ofvancomycin. A concentration of 100 μg/ml vancomycin cannot be achievedwith intravenous application.

2. Use of Different Antibiotics, i.e. Rifampin and Fosfomycin or theirCombination for the Treatment of Intracellular Infections

2.1. Use of Different Antibiotics, i.e. Rifampin and Fosfomycin or theirCombination for the Treatment of Intracellular Infections of OsteoblastsMG63 Infected with Staphylococcus aureus Subsp. aureus Rosenbach (ATTC6538P)

The experimental set up for determination of intracellular infection ofosteoblastic MG63 cells infected with S. aurerus ATTC 6538P wasessentially the same as above.

However, in order to eliminate extracellular S. aurerus ATTC 6538P eachcell culture was treated with lysostaphin after infection before addingthe antibiotics.

For this purpose the bacterial suspension was removed from each well andthe cells were washed once with warm DMEM containing 10% FCS. 250 μg/ml25 μg/ml lysostaphin solution was added to each well. The cells wereincubated for 10 min at 37° C. Afterwards no extracellular bacteriacould be observed microscopically. The lysostaphin solution was removedcompletely and the cells were washed once with 1 ml warm DMEM.Afterwards the antibiotic solutions having the following compositionswere added:

-   -   100 μg/ml vancomycin    -   100 μg/ml gentamicin    -   0.01-100 μg/ml rifampin    -   10-1000 μg/ml fosfomycin    -   1 μg/ml rifampin+100 μg/ml fosfomycin    -   1 μg/ml rifampin+100 μg/ml gentamicin    -   1-100 μg/ml moxifloxacin        The infected cells were incubated for 24 h at 37° C. under CO₂        atmosphere.

In order to determine the metabolic activity of osteoblastic MG63 cellsafter infection, the cell supernatant was removed and 1 ml of warm freshcell culture medium was added to each well. Afterwards 200 μl of MTTsolution (3[4,5-Dimethylthiazol-2-yl]-2,5-diphenaltetrazoliumbromide)was added to each well. The cultures were incubated for 2 h at 37° C.under 5% 00 ₂ atmosphere. The cell culture supernatant was removed andthe formazan, which was formed due to metabolic activity, wassolubilised with 1 ml isopropanol. 200 μl of each suspension weretransferred to a 96 well microtiter plate and the absorbance at 540 nmwas measured with an ELISA reader (Tecan).

The absorbance at 540 nm is an indicator for the metabolic activity ofthe osteoblastic MG63 cells. The intracellular propagation of thecytotoxic S. aurerus strain ATTC 6538P in osteoblastic MG63 cells leadsto the death of the infected cell. The lower the extinction is the loweris the metabolic activity of the cells and thus the stronger is theinfection of the cells with S. aurerus ATTC 6538P.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

FIGS. 3 a-d show he influence of the different antibiotics on themetabolic activities of osteoblastic MG63 cells

Vancomycin is unable to penetrate into the cell and thus does notinfluence the intracellular propagation of S. aurerus inside theosteoblastic cells. Therefore, the metabolic activity of theosteoblastic cells is strongly reduced due to the infection with S.aurerus ATTC 6538P (FIGS. 3a-d ). The same applies to gentamicin.Nevertheless, in combination of gentamicin with rifampin the metabolicactivity was higher than for rifampin alone (data not shwon).

Rifampin on the other hand is able to reduce the cell death caused by S.aurerus ATTC 6538P drastically (FIG. 3a ). Already small concentrations(0.006 μg/ml) are sufficient in increasing the metabolic activity.

Fosfomycin also influences the intracellular propagation of S. aurerusATTC 6538P and thus the metabolic activity of the infected osteoblasticcells (FIG. 3b ). 10 μg/ml fosfomycin increases the metabolic activityonly slightly, whereby 100 μg/ml had the maximal effect and almostdoubled the metabolic activity. This result is surprising since so farit has not been known that fosfomycin is be able to penetrate intocells. It is only known that fosfomycin can penetrate into neutrophils.

The combination of rifampin and fosfomycin also leads to an increase ofmetabolic activity (FIG. 3d ), even showing a synergistic effect (FIG.3c ).

Also the application of 1 to 100 μg/ml moxifloxacin can inhibitintracellular growth of S. aurerus ATTC 6538P and thus increase themetabolic activity up to more than two fold (FIG. 3e ).

2.2. Use of Rifampin, Fosfomycin or their Combination for the Treatmentof Intracellular Infections of Osteoblasts MG63 Infected withStaphylococcus aureus Subsp aureus (BAA44)

The experimental set up for determination of intracellular infection ofosteoblastic MG63 cells infected with S. aurerus BAA44 was essentiallythe same as above described for S. aurerus ATTC 6538P,

Because the non-cytotoxic S. aurerus BAA44 persists in osteoblasts anddoes not divide intracellularly like the cytotoxic strain S. aurerusATTC6538P, the intracellular localisation of S. aurerus BAA44 does notresult in cell death of the osteoblastic cells. The intracellularinfection of the osteoblastic MG63 cells with S. aurerus BAA44 couldtherefore not be determined on basis of the metabolic activity of thecells and was determinded via cell lysis and counting of theintracellular CFU instead.

Antibiotic solutions having the following compositions were added:

-   -   100 μml vancomycin    -   2.5-40 μg/ml rifampin    -   25-400 μg/ml fosfomycin    -   and their mixtures in different ratios as given below.        The infected cells were incubated with the antiobioics for 24 h        at 37° C. under 5% CO₂ atmosphere.

Afterwards the cells are washed once with PBS pH 7.4 (phosphate buffersolution) followed by lysis with 1 ml 0.1% Triton X100 in ringer'ssolution. The lysates were treated for 5 min with ultrasound. Thelysates are thoroughly resuspended with a pipette. Only one 24 wellplate was handled and the other plates were stored at 4° C. in order tominimize bacterial growth in the lysate. 100 μl lysate were undilutedstreaked out on Caso agar plates, incubated over night at 37° C. and thecolonies were counted.

FIGS. 4a-4g show the CFU value per well as an indicator for the degreeof intracellular S. aurerus BAA44 infections of osteoblastic MG63 cells.The lower the CFU value is the lower is the infection rate of theosteoblastic cells with S. aurerus BAA44. This correlates to theefficacy of the added antibiotic. Due to the weak intracellular growthof S. aurerus BAA44, a decrease in CFU is caused by the bactericidialeffect of the antibiotics.

Fosfomycin in concentration between 50-400 μg/ml shows a good efficacyon the infection rate with intracellular located S. aurerus BAA44 (FIG.4a ). Surprisingly the effect of fosfomycin can be achieved atconcentrations allowing for intravenous application (100-400 μg/ml,preferably 132-297 μg/ml in serum). Because of its excellent tissuepenetration high fosfomycin concentrations are also achieved in bone.Therefore, fosfomycin is successfully applied in the treatment ofosteomyelitis

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

Athough S. aurerus BAA44 is a rifampin resistant strain rifampin shows agood intracellular potency (FIG. 4b ).

Rifampin and fosfomycin show clearly a synergistic effect in varyingconcentration ratios (FIG. 4c-g ).

Even in case of the rifampin resistant S. aurerus BAA44 the appliedconcentrations were sufficient enough in order to allow a systemictreatment of bone infections. Therefore, the combination of rifamycinand fosfomycin is suitable for treating osteomyelitis and can also beapplied systemically.

3. Use of a Combination of Rifampin and Fosfornvcin as Anti-Adhesives onSurfaces of Medical Substrates

3.1. Adhesion of Staphylococcus aureus Subsp. aureus Rosenbach (ATTC6538P) on a Titanium Substrate Coated with Rifampin and Fosfomycin

An overnight culture of S. aurerus ATTC 6538P was prepared by infecting5 ml Caso-Bouillon medium with S. aurerus ATTC 6538P. The cultures wereincubated with shaking (450 U/min) over night at 37° C. 100 μl of theovernight cultures were transferred into 5 ml Caso Boulillon medium andincubated for 2 h at 37° C. with shaking (450 U/min). The bacterialdensity was determined photometrically. The bacterial suspension wasdiluted 1:2 in Caso Bouillon prior measurement. A bacterial suspensionwith a density of 1×10⁵ CFU/ml in Caso Boullion with 10% FCS was usedfor the adhesion experiments.

Differently coated 2 cm titanium discs were used as samples:

-   -   titanium discs sand blasted as negative control,    -   titanium discs sand blasted and coated with 200 μg/cm²        vancomycin,    -   titanium discs simultaneously coated with 50 μg/cm² rifampin and        200 μg/cm² fosfomycin calcium,    -   titanium discs coated in a first step with 50 μg/cm² rifampin        and in a second step with 200 μg/cm²fosfomycin calcium, and    -   titanium discs coated in a first step with 200 μg/cm²fosfomycin        calcium and in a second step with 50 μg/cm² rifampin.

After coating the titanium discs were washed either one time or threetimes with PBS. The coated and uncoated titanium discs were incubatedfor 5 min at room temperature with 5 ml PBS. This was repeated two moretimes. In the third circle the titanium discs were incubated for 1 h atroom temperature. Before removing the PBS solution the titanium discswere turned or swivelled in order to increase the detachment of theantibiotics. Afterwards the titanium discs were transferred into sterile12 well plates.

The different titanium samples were incubated with 2 ml bacterialsuspension for 1.5 h at 37° C. without shaking.

Afterwards the bacterial suspension was removed and the discs werewashed three times with 2.5 ml PBS. After the last washing cycle eachdiscs was placed in 10 ml sterile ringer's solution. Only one disc ofeach group was simultaneously examined while the other discs were storedat 4° C. The titanium discs in the ringer's solution were exposed toultra sound for 10 min in order to detach the adhered bacteria. Thesuspensions comprising the detached bacteria were diluted (1:10, 1:100)and streaked out on a Caso agar plate. The agar plates ere incubatedover night at 37° C. and the next day the colonies were counted.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

FIG. 5a shows the CFU of S. aurerus ATTC 6538P per titanium discafterincubation for 1.5 h with the bacteria. Vancomycin had only ananti-adhesive effect after one washing step, but did not reveal anyanti-adhesive effect after three washing steps. In fact, the number ofS. aurerus cells adhered to the vancomycin coated discs was identicaltothe uncoated discs. However, the combination rifampin and fosfomycinshowed a strong anti-adhesive effect. The effect depended only slightlyon the number of washing steps. Obviously, the rifampin/fosfomycincoating was less likely to be removed completely from the titaniumsurface by several washing steps than vancomycin.

The order of coating the discs with rifampin and fosfomycin—together,first rifampin then fosfomycin; first fosfomycin then rifampin—does notseem to influence the effect (FIG. 5a ).

3.2. Adhesion of Staphylococcus epidermis ATTC 35984 on a TitaniumSubstrate Coated with a Combination of Rifampin and Fosfomycin

The experimental set up was essentially the same as described above forS. aurerus ATTC 6538P.

Differently coated 2 cm titanium discs were used as samples:

-   -   titanium discs (sand blasted) as negative control,    -   titanium discs coated in a first step with 50 μg/cm² rifampin        and in a second step with 200 μg/cm²fosiomycin calcium, and    -   titanium discs coated in a first step with 200 μg/cm²fosfomycin        calcium and in a second step with 50 μg/cm² rifampin.

The coated titanium discs were washed three times with 5 ml PBS beforeincubation with S.epidermis.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

The experimental results for S.epidermis (FIG. 5b ) support the resultsfound in case of S. aurerus ATTC6538P. The adhesion of S.epidermis ATTC35984 on uncoated titanium was lower than the adhesion of S. aurerusATTC6538P. This can relate to the fact that S.epidermis preferablyattaches to plastics or hydroxyapatite but less to titanium. Althoughthe titanium discs were washed three times before incubation with thebacteria, the combination rifampin and fosfomycin shows a stronganti-adhesive effect.

3.3. Adhesion of Staphylococcus aureus BAA44 on a Titanium SubstrateCoated with a Combination of Rifampin and Fosfomycin

The experimental set up was essentially the same as described above forS. aurerus ATTC 6538P.

The following coated 2 cm titanium discs were used as samples:

-   -   titanium discs sand blasted as negative control,    -   titanium discs coated with 200 μg/cm² vancomycin    -   titanium discs sand blasted and coated in a first step with 50        μg/cm² rifampin and in a second step with 200 μg/cm² fosfomycin        calcium.

The coated titanium discs were washed either once or twice with 5 ml PBSbefore incubation with S. aurerus BAA44 for 1.5 h.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

FIG. 5c shows the CFU on the discs after incubation with S. aurerusBAA44 . Vancomycin reduced the adhesion of S. aurerus BAA44 only if thedisc were washed once: After two washing steps all vancomycin seems tobe removed and no reduction of bacterial adhesion could be observed.Despite the rifampin-resistance of S. aurerus BAA44 the combinationrifampin/fosfomycin had a strong anti-adhesive effect, which was onlyslightly diminished if the discs were washed twice instead of oncebefore incubation with the bacteria.

3.4. Bactericidal Activity of Titanium Substrate Coated with Rifampinand Fosfomycin Against S. aurerus BAA44

An overnight culture of S. aurerus BAA44 was prepared by infecting 5 mlCaso-Bouillon medium with S. aurerus BAA44. The cultures were incubatedwith shaking (450 U/min) over night at 37° C. 100 μl of the overnightcultures were transferred into 5 ml Casio Boulillon medium and incubatedfor 2 h at 37° C. with shaking (450 U/min) prior to the incubation withthe titanium discs. The bacterial density was determinedphotometrically.

A bacterial suspension with a density of 1×10⁴ CFU/ml in Minimal Medium(PBS, 0.2% ammonium chloride, 0.2% sodium sulphate, 0.25% glucose, 1%Caso Bouillon, 50 μg/ml glucose-6-phosphate) was used in the adhesionassay. The Minimal Medium was used instead of Casa Bouillon to minimizethe bacterial growth.

Differently coated 2 cm titanium discs were used as samples:

-   -   titanium discs (sand blasted) as negative control,    -   titanium discs (sand blasted) coated with 200 μg/cm² vancomycin,    -   titanium discs coated in a first step with 300 μg/cm²fosiomycin        calcium and in a second step with 70 μg/cm² rifampin.

After coating, the titanium discs were washed three times with 2.5 mlPBS at room temperature.

The different titanium samples were incubated with 2 ml bacterialsuspension for 15.5 h at 37° C. without shaking.

Afterwards the CFU in the supernatant as well as the adhered bacteria onthe titanium discs were analysed. The supernatant was diluted 1:10 inPBS, 100 μl of the dilution were streaked out on Caso agar plates. Thediscs were washed four times with 2.5 ml PBS to remove not adherentbacteria. After the last washing cycle each discs was placed in 10 mlsterile ringer's solution. Only one disc of each group wassimultaneously examined while the other discs were stored at 4° C. Thetitanium discs in the ringer's solution were exposed to ultra sound for10 min in order to detach the adhered bacteria. The suspensionscomprising the detached bacteria were diluted (1:10, 1:100, 1:1000) andstreaked out on a Caso agar plate. The agar plates were incubated overnight at 37° C. and the next day the colonies were counted.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

The results are shown in FIG. 5d . The bacterial growth in the negativecontrols was reduced by using Minimal Medium, but nevertheless thebacterial CFU in the supernatant increased ten times during theincubation period. Surprisingly, more CFU could be found adhered to theuncoated disc than in the supernatant.

Vancomycin reduced the bacterial growth in the cell culture supernatantcompared to the uncoated control slightly, but the adherence of thebacteria was even more reduced. However, vancomycin could not exhibitany bactericidal effect and more than 50,000 CFU could be found on thevancomycin coated titanium samples.

Although the titanium discs were washed three times before the adhesionassay, the fosfomycin/rifampin combination displayed a dear bactericidalactivity against the rifampin-resistant strain BAA44. No CFU could bedetected in the supernatant and less than 100 CFU adhered to thetitanium surface. This corresponds to a 86,000-fold reduction inbacterial adherence compared to uncoated titanium and a 470-foldreduction compared to the vancomycin coating.

It was expected that a soluble antibiotic coating without carrier matrixe.g. polymer matrix unfolds its efficacy by dissolving into the tissuefluid after implantation. The colonization of the implant or prosthesisis then hampered by killing the planktonic bacteria before colonizationand reduction of bacterial propagation due to the efficacy of thedissolved antibiotics. After several washing steps the amount ofrifampin and fosfomycin left on the discs, and thus available in thesupernatant, was still high enough for showing antibacterial efficacy inthe supernatant. Therefore, the rifampin/fosfomycin coating is stableenough to get in contact with tissue fluids and blood duringimplantation and is still effective in preventing bacterial adherence tothe implant surface and the surrounding tissue. This property isespecially important for staphylococci infections, because staphylococcido not adhere exclusively to implants but to the extracellular matrix oftissue as well.

4. Use of Rifampin and Daptomycin or their Combination for Treatment ofAcute Infection of Osteoblasts MG63 Cells with Staphylococcus aureusSubsp. aureus (BAA44)

Osteoblastic MG63 cells were detached with the cell detachment mediumAccutase 24 hours before infection. The cell number was determined usingthe Neubauer counting chamber. Cells were seeded onto uncoated 24wellplates with a cell density of 1.5×10⁴ cells/cm² in 1 ml DMEM (Dulbecco'sModified Eagle's Medium) with 10% FCS (fetal calf serum), 1% Glutamax-Iand 1% Natrium Pyruvat and incubated at 37° C. and 5% CO₂.

An overnight culture of S. aurerus BAA44 was prepared by infecting 5 mlCaso-Bouillon medium with S. aurerus BAA44. The cultures were incubatedwith shaking (450 U/min) over night at 37° C. 100 μl of the overnightcultures were transferred into 5 ml Caso Boulillon medium and incubatedfor 2 h at 37° C. with shaking (450 U/min) prior to infection.

The cell culture supernatant of the osteoblastic MG63 cells was removedwith a pipette from the wells. 1 ml containing 1×10⁶ S. aurerus BAA44CFU was added to each well containing also antibiotics having thefollowing compositions:

-   -   50 μg/ml vancomycin    -   2.5 μg/ml rifampin    -   1.25-10 μg/ml daptomycin    -   and their mixtures in different ratios as given below.

The combined osteoblastic cells, bacteria, and antibiotic compositionswere incubated for 18 h at 37° C. under 5% CO₂ atmosphere.

Afterwards the cells were washed once with PBS pH 7.4 (phosphate buffersolution) followed by lysis with 1 ml 0.1% Triton X100 in ringer'ssolution. The lysates were thoroughly resuspended with a pipette. Onlyone 24 well plate was handled and the other plates were stored at 4° C.in order to minimize bacterial growth in the lysate. The lysates werediluted 1:10 in PBS, 100 μl of diluted lysate were streaked out on Casoagar plates, incubated over night at 37° C. and the colonies werecounted.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

Because the cells were not treated with lysostaphin after infection, theCFU value per well (FIG. 6) is an indicator for the degree ofintracellular infection of osteoblastic MG63 cells with S. aurerus BAA44as well as for S. aurerus BAA44 adhered extracellularly to osteoblasticMG63 cells. The lower the CFU value is the lower is the infection rateof the osteoblastic cells with S. aurerus BAA44. This correlates to theefficacy of the added antibiotic.

Because the strain is rifampin-resistant the effect of 2.5 μg rifampinwas less than for vancomycin and daptomycin, but overgrowth of the MG63cells with planktonic S. aurerus BAA44 was prevented efficacious (datanot shown)

Daptomycin alone showed good efficacy already in concentrations of 1.25μg/ml and 2.5 μg/ml, whereas 5 μg/ml and 10 μg/ml could eradicate theinfection completely.

Despite the ineffectiveness of rifampin alone, the combination 2.5 μg/mlrifampin and 1.25 μg/ml or 2.5 μg/ml daptomycin respectively wassynergistic in eliminating all intracellular and extracellular adheredbacteria.

Because vancomycin is only weak bactericidal a very high concentrationof vancomycin was used in this experiment to increase its efficacy. Thisconcentration can never be achieved by intravenous application ofvancomycin. However, several hundred S. aurerus could escape vancoymicinby invading the osteoblastic cells, a phenomenon that has relevance invivo especially in the treatment of bone infections.

Daptomycin is in contrast to glycopeptides like vancomycin rapidlybactericidal and the bactericidal activity is concentration dependent.Therefore the higher concentrations of 5 and 10 μg/ml could eliminateall bacteria before they were able to invade the osteoblastic cells.

Local application of rifampin and daptomycin could be an efficienttreatment for acute bone infections. Daptomycin eliminates in highconcentrations very efficiently all extracellular bacteria and thusprevents infection of new osteoblasts, while rifampin is able toeradicate intracellular infected osteoblasts.

5. Coated or Impregnated Substrates for Medical Purposes

Rifampin was diluted in methanol in a concentration of 30-40 mg/ml.Fosfomycin calcium was suspended in ultrapure water in a concentrationof 100-140 μg/ml. No further additives were used. The titaniumendoprosthesis with different surface modifications (sand-blasted,porous coated, or hydroxyapatite coated) was coated directly with theantibiotic solutions using the ink-jet or the spray coating process. Thesurface can be coated with rifampin first, followed by fosfomycincalcium, the other way around, or both antibiotics simultaneously. Theresulting covering density was 50-70 μg/cm² rifampin and 300-350 μg/cm²fosfomycin.

Rifampin, fosfomycin disodium, and fosfomycin calcium were incorporatedinto collagen fleeces during the production process of the fleeces.Rifampin and fosfomycin disodium were added dissolved in acidifiedbuffer, while fosfomycin calcium was added in watery suspension. Thefinal concentrations were 0.1-0.2 mg rifampin per cm² collagen fleeceand 0.5 mg-2 mg fosfomycin per cm² collagen fleece, whereas fosfomycindisodium and fosfomycin calcium could contribute in varying proportionsto the final concentration of fosfomycin.

Rifampin and fosfomycin disodium were mixed with two different polymerson PMMA basis, zirconium dioxid, and glycine. Rifampin was added in anamount of 0.5-1.5% of the total weight, while fosfomycin disodium wasadded in an amount of 2.5-7.5% of the total weight. Thepolymer/antibiotic mixture was heated to 160-180° C. and PMMA beads weremanufactured directly on metal wires by injection moulding.

The person skilled in the art will recognize that the above givendescription is just one possibility out of many alternatives.

Numerous modifications and variations of practicing the presentinvention are possible in light of the above teachings and thereforewill fall within the scope of the following claims.

1. A pharmaceutical composition for use in the local treatment andprevention of a tissue infection, the pharmaceutical compositioncomprising a rifamycin and fosfomycin or a rifamycin and daptomycin in acoating, wherein the coating comprises 10 to 1000 μg/cm² antibiotics. 2.The composition according to claim 1, characterized in that therifamycin is rifampin, rifabutin, rifapentine or rifamixin.
 3. Thecomposition according to claim 1, characterized in that, saidcomposition comprises rifampin and fosfomycin.
 4. The compositionaccording to claim 1, characterized in that said composition furthercomprises a biofilm formation inhibitor.
 5. The composition according toclaim 4, characterized in that the biofilm formation inhibitor comprisessalicylic acid or a pharmaceutically acceptable derivative or saltthereof.
 6. The composition according to claim 1 characterized in thatthe infected tissue to be treated is acutely or chronically infected. 7.The composition according to claim 1, characterized in that saidcomposition is used in the treatment of extracellular and/orintracellular microbial infected tissue cells and/or for the preventionof extracellular and/or intracellular microbial infections of tissuecells.
 8. The composition according to claim 7, characterized in thatthe infected tissue cells are soft tissue cells and/or bone tissuecells.
 9. The composition according to claim 8, characterized in thatthe infected tissue soft tissue cells and/or bone tissue cells areosteoblasts, leucocytes, erythrocytes, keratinocytes, fibroblasts, fatcells, muscle cells and/or endothelial cells.
 10. Composition accordingto claim 7, characterized in that the tissue infection is caused bygram-negative and/or gram-positive bacteria, preferably by theStaphyloccoci type, most preferably by Staphylococcus aureus.
 11. Thecomposition according to claim 1, characterized in that said compositioncomprises rifamycin and fosfomycin in such a concentration that therifamycin reaches a concentration of 0.005 to 100 μg/ml, preferably0.006 to 80 μg/ml, most preferably 0.0075 to 10 μg/ml at the site to betreated, and fosfomycin reaches a concentration of 1 to 1000 μg/ml,preferably 5 to 800 μg/ml, most preferably 10 to 200 μg/ml fosfomycin atthe site to be treated.
 12. The composition according to claim 1,characterized in that said composition comprises rifamycin anddaptomycin in such a concentration that the rifamycin reaches aconcentration of 0.005 to 100 μg/ml, preferably 0.006 to 80 μg/ml, mostpreferably 0.0075 to 20 μg/ml at the site to be treated, and daptomycinreaches a concentration of 0.1 to 100 μg/ml, preferably 0.5 to 80 μg/ml,most preferably 1 to 20 μg/ml at the site to be treated.
 13. Thecomposition according to claim 1, characterized in that the coatingcomprises a substrate arranged and provided for being used as carrier ofsaid composition, in particular for local antibiotic therapy of an acuteor chronic infection of a tissue of a subject.
 14. Composition accordingto claim 13, characterized in that said substrate comprises a fleece, afabric, a polymethyl methacrylate, a copolymer of methylmethacrylate andmethylacrylate, a biodegradable polymer, polyethylene, a metal, aceramic, a bone cement, a bone substitute, or a combination of any ofthe foregoing.
 15. The composition according to claim 14, characterizedin that the fleece or the fabric comprises a natural or synthetic fibre,particularly polylactide, and/or collagen.
 16. The composition accordingto claim 13, characterized in that said substrate comprises animplantable prosthesis, in particular a hip prosthesis, a shoulderprosthesis, an elbow prosthesis, a knee prosthesis, a vertebral implant,or an implant for trauma surgery.